MECHANISM OF B LYMPHOCYTE SOMATIC HYPERMUTATION

这个医生科学家补助金提案的长期目标是发展 了解B淋巴细胞 进行调节免疫球蛋白（IG）基因体细胞超突变。 这 该过程允许产生更大的抗体多样性和发展 更有效的高亲和力抗体。 以前的研究尝试 这一过程的分子机制和详细组织 由于缺乏合适的B细胞模型，这一过程受到阻碍。 存在重链可变区高频率突变的证据， 18.81亚克隆的链IG基因，Abelson转化的鼠前B 细胞系 在这个项目的第一阶段，除了课程， 分子生物学，将确认18.81细胞系 在变量（V）中经历频繁和自发的点突变 其内源性免疫球蛋白基因的区域，并且V 区域突变大于恒定区突变。 实际 将建立血清学和分子测定法来鉴定突变体 基因. 将比较突变的速率和性质 发生在转染和内源性IG基因中。 在第二阶段， 将使用IG基因的转染构建体来确定DNA 所需的侧翼和/或编码序列中的序列 突变的发生，看看是否有特定的序列是针对这些 事件 这些序列被证明是重要的这一进程将是 通过定点诱变进行分析，并将用于搜索 通过凝胶阻滞参与调节这一过程的蛋白质 比色法 将使用转基因小鼠检查它们在体内的相关性。 这些蛋白质的基因将被克隆以确定序列， 表达，并在此体外系统中。 机理的确定 对这一过程的监管将解决一个主要的机制， 来产生抗体多样性。 它还将允许产生 更高亲和力的单克隆抗体，例如用于抗- 肿瘤或感染过程以及用于诊断目的。 突变 在IG基因中的表达在恶性肿瘤的发生中是重要的， 伯基特淋巴瘤。 对这一过程的更好理解可能会导致 治疗这些和其他淋巴肿瘤的新方法， 淋巴细胞分化的过程。

The long term goal of this physician-scientist grant proposal is to develop the skills required to understand the mechanisms by which B lymphocytes undergo and regulate immunoglobulin (Ig) gene somatic hypermutation. This process allows for generation of greater antibody diversity and development of more effective higher affinity antibodies. Previous attempts to study the molecular mechanisms and detailed organization of this process have been hampered by the lack of an appropriate B cell model for this process. Evidence exists for high frequency mutations of the variable region heavy chain Ig gene of a subclone of 18.81, an Abelson-transformed murine pre-B cell line. During Phase I of this project, in addition to coursework in molecular biology, will be the confirmation that the 18.81 cell line undergoes frequent and spontaneous point mutations in the variable (V) region of its endogenous immunoglobulin gene and that the frequency of V region mutation is greater than constant region mutation. Practical serological and molecular assays will be established to identify mutant genes. Comparisons will be performed of the rate and nature of mutations that occur in transfected and endogenous Ig genes. In Phase II, transfected constructs of an Ig gene will be used to determine DNA sequences in the flanking and/or coding sequences which are required for mutations to occur and to see if specific sequences are targeted for these events. Those sequences shown to be important for this process will be analyzed by site directed mutagenesis and will be used to search for proteins involved in the regulation of this process by gel retardation assay. Their relevance in vivo will be examined using transgenic mice. Genes for these proteins will be cloned for determination of sequence, expression, and in this in vitro system. Determination of the mechanism and regulation of this process would resolve one of the major mechanisms for generating antibody diversity. It would also allow the generation of higher affinity monoclonal antibodies such as those used for anti- neoplastic or infectious processes and for diagnostic purposes. Mutations in the Ig gene are important in the oncogenesis of malignancies such as Burkitt's lymphoma. Improved understanding of this process may lead to novel treatments for these and other lymphoid tumors, and for manipulation of lymphoid cell differentiation.